In the previous support period, we developed tools for continuous magnetic flow cell sorting based on cell magnetophoresis. We also developed cell tracking velocimetry to measure cell magnetophoretic mobility. Cell sorting based on magnetophoresis of freely suspended cells has the potential for improved performance over current magnetic separation methods that rely on cell immobilization on magnetic wire and bead matrices. By optimizing both the flow sorter channel geometry and the applied magnetic field (annular flow in a quadrupole field), we achieved high sorting speed (10[7] cells/s) with high cell purity (90% purity of CD34+, at recovery of 30%). Furthermore, separated cell viability remained high due to relatively low shear stress experienced during sorting. The technology development was aided by collaboration with the Cleveland Clinic Taussig Cancer Center and the Ohio State University. Based on the strengths of the current effort, we propose to further evaluate the technology with the focus on the following: Aim 1: To compare the technology with a benchmark high-gradient magnetic separation (HGMS) method based on extrinsic cell magnetization with antibody-conjugated magnetic nanoparticles. First, we will test positive cell separation of long term colony-initiating cells from committed blood progenitor cells. The fractionated cells will be fully characterized in terms of their phenotypic and functional characteristics (in collaboration with Dr. Maciejewski at the Taussig Cancer Center). Second, we will test negative separation in application to isolation of rare cancer cells from circulating blood. Circulating white blood cells will be targeted by anti pan WBC antibody (such as anti CD45 Ab) and will be separated from the un-labeled, non-mobile non-WBCs, representing the likely candidates for the cancer cells in the circulating blood (in collaboration with Dr. Chalmers at the OSU). Aim 2: To investigate the intrinsic magnetophoresis of fresh, un-manipulated (unlabeled) cells. Magnetophoretic cell sorting may allow separation of cells differing in their intrinsic magnetization (without application of extrinsic magnetic nanoparticles). We will test the technology first on cell lines that can be induced to express high levels of ferritin, and second, on selected primary leukemia samples known for their elevated intracellular ferritin level. Aim 3: To further investigate the magnetophoretic process and refine the cell tracking velocimetry instrumentation for applications to cancer therapy. In particular, we will determine the applicability of MRI contrast agents to modification of the magnetic susceptibility of suspending medium to enhance magnetophoretic cell sorting and achieve high purity and recovery of the target cells (in collaboration with Dr. Hafeli). The proposed research will help to fully characterize the magnetophoretic cell sorting process, further improve the tools, and determine the applications relevant to the diagnosis and therapy of cancer.